Sheet tissue products

ABSTRACT

The present disclosure provides a tissue product comprising a tissue sheet. The sheet can be formed from a plurality of tissue matrix fragments. The fragments can have a length between about 5 μm and 300 μm, wherein the tissue matrix fragments are joined to one another to form the tissue sheet. In addition, methods of producing the tissue products and products made by the methods are provided.

This application claims priority under 35 U.S.C. §119 to U.S. Provisional Application No. 61/577,721, which was filed on Dec. 20, 2011.

The present disclosure relates to tissue products and, more particularly, to tissue products formed as elongated sheets.

Various tissue products have been produced to replace, augment, or treat tissue defects. For example, to replace or augment tissue defects, acellular dermal tissue matrices such as ALLODERM® and STRATTICE™, which are available from LIFECELL® Corporation (Branchburg, N.J.), may be used.

Although suitable for certain applications, further improvements in the ability of tissue products to be used for soft or hard tissue treatment are desirable. Accordingly, the present disclosure provides improved tissue products produced from tissue matrices.

SUMMARY

According to certain embodiments, a tissue product is provided. The product can include a sheet of material, wherein the sheet comprises a plurality of tissue matrix fragments having a length between about 5 μm and 300 μm, wherein the tissue matrix fragments are joined to one another to form the tissue sheet.

According to certain embodiments, a method for producing a tissue treatment composition is provided. The method can include selecting a tissue matrix and treating the tissue matrix to produce fragments having a length between about 5 μm and 300 μm. The method can further include forming the fragments into a sheet; and treating the sheet to join the fragments to one another.

According to certain embodiments, a method of treating a tissue site is provided. The method can include selecting a tissue site and selecting a tissue product comprising a sheet of material, wherein the sheet comprises a plurality of tissue matrix fragments having a length between about 5 μm and 300 μm, wherein the tissue matrix fragments are joined to one another to form the tissue sheet. The method can further include placing the tissue sheet in or on the tissue site.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a process for producing a tissue product according to various embodiments.

DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS

Reference will now be made in detail to certain exemplary embodiments according to the present disclosure, certain examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts.

In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Any range described herein will be understood to include the endpoints and all values between the endpoints.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.

As used herein “tissue product” will refer to any human or animal tissue that contains extracellular matrix proteins. “Tissue products” can include intact tissue matrices, acellular or partially decellularized tissue matrices, decellularized tissue matrices that have been repopulated with exogenous cells, and/or cellular tissues that have been processed to change the orientation of at least some of the collagen fibers within the tissue's extracellular matrix.

Various tissue products are available for treatment of hard and soft tissues. Such tissue products can include processed tissues, which have been treated to remove some or all of the cellular components and/or other materials (e.g., antigens and lipids). Such tissue products can be used for treatment, repair, regeneration, and/or augmentation of a variety of different tissues. For example, acellular tissue matrices can be used to replace soft tissue lost or damaged due to, for example, surgery, trauma, disease, and/or atrophy.

Current acellular tissue matrices or other tissue scaffold or replacements materials (e.g., processed collagen or synthetic materials) are available in a variety of different forms. STRATTICE™ and ALLODERM® (LIFECELL® Corporation, Branchburg, N.J.) are two acellular dermal tissue matrix products, that are sold as sheets. For example, STRATTICE™ and ALLODERM® can be used, for example, for soft tissue augmentation, e.g., to treat abdominal wall defects; and CYMETRA® can be injected for soft tissue augmentation.

According to certain embodiments, a tissue product is provided. The product can include a sheet of material, wherein the sheet comprises a plurality of tissue matrix fragments having a length between about 5 μm and 300 μm, wherein the tissue matrix fragments are joined to one another to form the tissue sheet.

According to certain embodiments, a method for producing a tissue treatment composition is provided. The method can include selecting a tissue matrix and treating the tissue matrix to produce fragments having a length between about 5 μm and 300 μm. The method can further include forming the fragments into a sheet; and treating the sheet to join the fragments to one another.

According to certain embodiments, a method of treating a tissue site is provided. The method can include selecting a tissue site and selecting a tissue product comprising a sheet of material, wherein the sheet comprises a plurality of tissue matrix fragments having a length between about 5 μm and 300 μm, wherein the tissue matrix fragments are joined to one another to form the tissue sheet. The method can further include placing the tissue sheet in or on the tissue site.

In certain embodiments, the tissue products produced as described herein provide improved properties when implanted or during storage. For example, the products described herein may be less susceptible to damage caused during freezing than other acellular tissue matrices. In addition, the matrices may have an improved ability to allow cellular ingrowth and vascularization.

FIG. 1 illustrates a process for producing a tissue product according to various embodiments. As shown at step 101, the process begins with selecting a tissue matrix 100. Suitable tissue matrices are discussed further below, but the tissue matrices can include any substantially acellular tissue matrix produced from human or animal tissue, which retains the ability to support cellular ingrowth and tissue regeneration without excessive inflammation. Certain exemplary tissue matrices that may be used include STRATTICE™ and ALLODERM® (LIFECELL® Corporation, Branchburg, N.J.), which are porcine and human acellular dermal matrices, respectively. However, other suitable tissue matrices can be used, including, for example, small-intestine submucosa. In addition, the tissue matrices can include intact tissues (not decellularized) and/or tissues that have been partially decellularized and/or populated with exogenous cells.

Next, as shown at step 111, the matrix 100 is processed to produce fragments 110. The tissue fragments 110 can be formed using a range of sizes and different morphologies. For example, in some embodiments, the tissue fragments 110 are in the form of small strands or threads of tissue matrix that has been treated to produce the desired size distribution and/or shape. In various embodiments, the strands or threads have a length between about 5 μm and 300 μm, between about 50 μm and 200 μm, between about 50 μm and 300 μm, or any values in between. In certain embodiments, the strands are approximately 40 microns×140 microns to 100 microns by 350 microns.

The tissue fragments 110 can be produced using a variety of processes. For example, any suitable cutting, grinding, milling, blending, shearing, or other mechanical process can be used, which produces the desired size and shape and does not cause unsuitable damage or change to the tissue matrix. In certain embodiments, the tissue fragments 110 are processed using a mill such as a SYMPAK® food mill or a QUADRO Attrition Mill (Quadro, Canada). In some embodiments, the tissue matrix 100 is cut into small pieces (e.g., 4 cm×4 cm) and then milled. In addition, the matrix may be blended briefly in a solution (e.g., PBS) prior to milling.

In some cases, the tissue matrices 100 can be processed to produce the fragments 110 when wet or submerged in a liquid. For example, the tissue matrices 100 can be milled or otherwise processed when submerged in a buffer such as PBS or any other suitable buffer. Further, after processing, the buffer can be at least partially removed by centrifuging or filtering to remove some or all of the liquid component. For example, a suitable centrifugation protocol can include centrifuging at 4,500 rpms for about 60 min.

After processing to produce tissue fragments 110, groups of the fragments 120 are formed to produce sheets 120 having a desired shape, as shown at Step 121. The specific shapes and sizes of the sheet 120 can vary based on the intended implantation site, to control space between particles to provide channels for cellular and vascular ingrowth, or to control the ability of the sheet to conform to related anatomy. The tissue sheets 120 can be shaped using a variety of molding or shaping processes. For example, the fragments 110 may be compressed using a rolling device such as a pasta/dough roller or similar device.

In some embodiments, the tissues are compressed with sufficient force to at least partially align fragments within the sheets. For example, the fragments may be rolled or compressed to align at least some of the along one axis of the sheet. Alternatively, the material may be rolled along one axis, folded, and then rolled along a different axis. The process may be repeated to produce partially aligned fragments, or to produce a sheet with random distribution of fragments (e.g., with no alignment).

After producing the desired shape, the fragments are joined to one another to form stable structures, as shown at Step 131. In certain embodiments, the fragments are joined without the use of substantial amounts of binder or adhesives. In addition in some embodiments, the fragments are dried using a process that is believed to join the fragments without significant cross-linking. For example, in some cases, the fragments may have frayed ends that interlock with one another. Further, in some embodiments, the fragments may bind to one another by non-covalent binding. As discussed elsewhere, the sheets may be dried using a process such as convective drying, and such processes can produce sheets having fragments that are joined to one another.

In some cases, the fragments are joined while being compressed. For example, as noted above, the sheets may be compressed so as to at least partially align the fragments. In some cases, a compressive force can be applied during the joining process, e.g., the sheets are compressed during drying. As such, the final sheet can form a stable structure wherein fragments are aligned.

In some embodiments, the fragments are joined to one another by cross-linking. Cross-linking can be accomplished using a number of processes such as dehydrothermal cross-linking, exposure to UV light, and/or chemical cross-linking. In some embodiments, a dehydrothermal cross-linking process is used to allow cross-linking while simultaneously drying the sheets. In addition, using any of the cross-linking processes, the sheets may be further dried (e.g., by freeze-drying or air drying) to remove additional moisture.

In various embodiments, the tissue products can be selected to have certain properties that facilitate implantation and tissue filling and/or regeneration. For example, the sheets can be selected such that they swell when contacted with an aqueous environment, as may be present in a tissue site. As such, the sheets can expand when implanted to fill a selected tissue site.

In some embodiments, the sheets are dried by convective heating. For example, frozen particles may be placed in a convection dryer (e.g., HARVEST Brand Kitchen Convection Dryer). Drying may be performed at approximately 45° C. However, lower or higher temperatures may be used, as long as temperatures that cause unacceptable denaturation or other tissue damage are not used. In addition, it should be noted, that even when partially or mostly dried, as described above using a panner, the particles may be further dried to remove excess moisture.

In various embodiments, sheets produced as described above can be assembled to produce products having various structures. For example, in some embodiments, multiple sheets are layered and joined to one another. In some cases, sheets having fragments aligned in a particular direction are stacked to produce a thicker sheet. In some cases, one or more of the sheets in the stack can be positioned such that the fragments are oriented in a direction different than the orientation of the fragments in another sheet. As such sheets having various mechanical properties (e.g., higher tensile strength and/or varying flexibility) can be produced.

After drying, the sheets are packaged and sterilized to form a final product 140, as shown at Step 141. The product can be package in a variety of known medical containers and can be sterilized using conventional processes as long as the processes do not damage the product (e.g., by excessive cross-linking) in an unacceptable manner. In some embodiments, the product can be packaged in foil-to-foil pouches and irradiated. In some embodiments, the product can be irradiated with e-beam radiation. Suitable e-beam doses can include 15-22 kGy or ranges therebetween.

The tissue products of the present disclosure can be used to treat a variety of different soft tissue or hard tissue sites. For example, the products can be used to replace, repair, regenerate or augment tissue lost or destroyed due to surgery, trauma, and/or any pathologic process. In some embodiments, the tissue products can be implanted in a soft tissue site such as a lumpectomy site. In other embodiments, the products can be used to treat or augment bone, muscle, subcutaneous tissue, and/or adipose tissue.

In certain embodiments, internal negative pressure can be applied within the tissue product. In certain embodiments, negative pressure can serve to draw cells from surrounding tissue into the implanted acellular tissue product, increasing the rate at which native cells migrate into the tissue product and enhancing the speed and/or overall effectiveness of tissue approximation.

In certain exemplary embodiments, internal negative pressure is delivered to the acellular tissue matrix by a reduced pressure therapy device. The reduced pressure therapy device can include a pump fluidly connected, e.g., through a fluid passage or tubing to the acellular tissue matrix, and which delivers reduced or negative pressure to the acellular tissue matrix. A variety of reduced pressure therapy devices can be used. For example, suitable reduced pressure therapy devices include V.A.C.® therapy devices produced by KCI (San Antonio, Tex.).

Acellular Tissue Matrices

The term “acellular tissue matrix,” as used herein, refers generally to any tissue matrix that is substantially free of cells and/or cellular components. Skin, parts of skin (e.g., dermis), and other tissues such as blood vessels, heart valves, fascia, cartilage, bone, and nerve connective tissue may be used to create acellular matrices within the scope of the present disclosure. Acellular tissue matrices can be tested or evaluated to determine if they are substantially free of cell and/or cellular components in a number of ways. For example, processed tissues can be inspected with light microscopy to determine if cells (live or dead) and/or cellular components remain. In addition, certain assays can be used to identify the presence of cells or cellular components. For example, DNA or other nucleic acid assays can be used to quantify remaining nuclear materials within the tissue matrices. Generally, the absence of remaining DNA or other nucleic acids will be indicative of complete decellularization (i.e., removal of cells and/or cellular components). Finally, other assays that identify cell-specific components (e.g., surface antigens) can be used to determine if the tissue matrices are acellular.

In general, the steps involved in the production of an acellular tissue matrix include harvesting the tissue from a donor (e.g., a human cadaver or animal source) and cell removal under conditions that preserve biological and structural function. In certain embodiments, the process includes chemical treatment to stabilize the tissue and avoid biochemical and structural degradation together with or before cell removal. In various embodiments, the stabilizing solution arrests and prevents osmotic, hypoxic, autolytic, and proteolytic degradation, protects against microbial contamination, and reduces mechanical damage that can occur with tissues that contain, for example, smooth muscle components (e.g., blood vessels). The stabilizing solution may contain an appropriate buffer, one or more antioxidants, one or more oncotic agents, one or more antibiotics, one or more protease inhibitors, and/or one or more smooth muscle relaxants.

The tissue is then placed in a decellularization solution to remove viable cells (e.g., epithelial cells, endothelial cells, smooth muscle cells, and fibroblasts) from the structural matrix without damaging the biological and structural integrity of the collagen matrix. The decellularization solution may contain an appropriate buffer, salt, an antibiotic, one or more detergents (e.g., TRITON X-100™, sodium deoxycholate, polyoxyethylene (20) sorbitan mono-oleate), one or more agents to prevent cross-linking, one or more protease inhibitors, and/or one or more enzymes. In some embodiments, the decellularization solution comprises 1% TRITON X-100™ in RPMI media with Gentamicin and 25 mM EDTA (ethylenediaminetetraacetic acid). In some embodiments, the tissue is incubated in the decellularization solution overnight at 37° C. with gentle shaking at 90 rpm. In certain embodiments, additional detergents may be used to remove fat from the tissue sample. For example, in some embodiments, 2% sodium deoxycholate is added to the decellularization solution.

After the decellularization process, the tissue sample is washed thoroughly with saline. In some exemplary embodiments, e.g., when xenogenic material is used, the decellularized tissue is then treated overnight at room temperature with a deoxyribonuclease (DNase) solution. In some embodiments, the tissue sample is treated with a DNase solution prepared in DNase buffer (20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 20 mM CaCl₂ and 20 mM MgCl₂). Optionally, an antibiotic solution (e.g., Gentamicin) may be added to the DNase solution. Any suitable buffer can be used as long as the buffer provides suitable DNase activity.

While an acellular tissue matrix may be made from one or more individuals of the same species as the recipient of the acellular tissue matrix graft, this is not necessarily the case. Thus, for example, an acellular tissue matrix may be made from porcine tissue and implanted in a human patient. Species that can serve as recipients of acellular tissue matrix and donors of tissues or organs for the production of the acellular tissue matrix include, without limitation, mammals, such as humans, nonhuman primates (e.g., monkeys, baboons, or chimpanzees), pigs, cows, horses, goats, sheep, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, or mice.

Elimination of the α-gal epitopes from the collagen-containing material may diminish the immune response against the collagen-containing material. The α-gal epitope is expressed in non-primate mammals and in New World monkeys (monkeys of South America) as well as on macromolecules such as proteoglycans of the extracellular components. U. Galili et al., J. Biol. Chem. 263: 17755 (1988). This epitope is absent in Old World primates (monkeys of Asia and Africa and apes) and humans, however. Id. Anti-gal antibodies are produced in humans and primates as a result of an immune response to α-gal epitope carbohydrate structures on gastrointestinal bacteria. U. Galili et al., Infect. Immun. 56: 1730 (1988); R. M. Hamadeh et al., J. Clin. Invest. 89: 1223 (1992).

Since non-primate mammals (e.g., pigs) produce α-gal epitopes, xenotransplantation of collagen-containing material from these mammals into primates often results in rejection because of primate anti-Gal binding to these epitopes on the collagen-containing material. The binding results in the destruction of the collagen-containing material by complement fixation and by antibody dependent cell cytotoxicity. U. Galili et al., Immunology Today 14: 480 (1993); M. Sandrin et al., Proc. Natl. Acad. Sci. USA 90: 11391 (1993); H. Good et al., Transplant. Proc. 24: 559 (1992); B. H. Collins et al., J. Immunol. 154: 5500 (1995). Furthermore, xenotransplantation results in major activation of the immune system to produce increased amounts of high affinity anti-gal antibodies. Accordingly, in some embodiments, when animals that produce α-gal epitopes are used as the tissue source, the substantial elimination of α-gal epitopes from cells and from extracellular components of the collagen-containing material, and the prevention of re-expression of cellular α-gal epitopes can diminish the immune response against the collagen-containing material associated with anti-gal antibody binding to α-gal epitopes.

To remove α-gal epitopes, after washing the tissue thoroughly with saline to remove the DNase solution, the tissue sample may be subjected to one or more enzymatic treatments to remove certain immunogenic antigens, if present in the sample. In some embodiments, the tissue sample may be treated with an α-galactosidase enzyme to eliminate α-gal epitopes if present in the tissue. In some embodiments, the tissue sample is treated with α-galactosidase at a concentration of 300 U/L prepared in 100 mM phosphate buffer at pH 6.0. In other embodiments, the concentration of α-galactosidase is increased to 400 U/L for adequate removal of the α-gal epitopes from the harvested tissue. Any suitable enzyme concentration and buffer can be used as long as sufficient removal of antigens is achieved.

Alternatively, rather than treating the tissue with enzymes, animals that have been genetically modified to lack one or more antigenic epitopes may be selected as the tissue source. For example, animals (e.g., pigs) that have been genetically engineered to lack the terminal α-galactose moiety can be selected as the tissue source. For descriptions of appropriate animals see co-pending U.S. application Ser. No. 10/896,594 and U.S. Pat. No. 6,166,288, the disclosures of which are incorporated herein by reference in their entirety. In addition, certain exemplary methods of processing tissues to produce acellular matrices with or without reduced amounts of or lacking alpha-1,3-galactose moieties, are described in Xu, Hui. et al., “A Porcine-Derived Acellular Dermal Scaffold that Supports Soft Tissue Regeneration: Removal of Terminal Galactose-α-(1,3)-Galactose and Retention of Matrix Structure,” Tissue Engineering, Vol. 15, 1-13 (2009), which is incorporated by reference in its entirety.

After the acellular tissue matrix is formed, histocompatible, viable cells may optionally be seeded in the acellular tissue matrix to produce a graft that may be further remodeled by the host. In some embodiments, histocompatible viable cells may be added to the matrices by standard in vitro cell co-culturing techniques prior to transplantation, or by in vivo repopulation following transplantation. In vivo repopulation can be by the recipient's own cells migrating into the acellular tissue matrix or by infusing or injecting cells obtained from the recipient or histocompatible cells from another donor into the acellular tissue matrix in situ. Various cell types can be used, including embryonic stem cells, adult stem cells (e.g. mesenchymal stem cells), and/or neuronal cells. In various embodiments, the cells can be directly applied to the inner portion of the acellular tissue matrix just before or after implantation. In certain embodiments, the cells can be placed within the acellular tissue matrix to be implanted, and cultured prior to implantation. 

What is claimed is:
 1. A tissue sheet product, comprising: a first sheet of material, wherein the first sheet of material comprises a plurality of elongated tissue matrix fragments having a length between 5 μm and 300 μm, wherein at least a portion of the plurality of elongated tissue matrix fragments include frayed proximal and distal ends; and wherein the plurality of elongated tissue matrix fragments are joined to one another at the frayed proximal and distal ends to form the first sheet of material.
 2. The tissue sheet product of claim 1, wherein the plurality of elongated tissue matrix fragments are joined by convective drying.
 3. The tissue sheet product of claim 1, wherein the plurality of elongated tissue matrix fragments are cross-linked to one another.
 4. The tissue sheet product of claim 3, wherein the plurality of elongated tissue matrix fragments are cross-linked to one another using a dehydrothermal crosslinking process.
 5. The tissue sheet product of claim 1, wherein the plurality of elongated tissue matrix fragments are produced from acellular tissue matrix.
 6. The tissue sheet product of claim 1, wherein the first sheet of material swells when contacted with water.
 7. The tissue sheet product of claim 1, wherein the first sheet of material forms a porous structure with open channels that can support cellular ingrowth and vascularization.
 8. The tissue sheet product of claim 1, wherein the plurality of elongated tissue matrix fragments are elongated strands of tissue matrix.
 9. The tissue sheet product of claim 1, wherein each of the plurality of elongated tissue matrix fragments is a dermal tissue matrix.
 10. The tissue sheet product of claim 1, wherein each of the plurality of elongated tissue matrix fragments is a porcine tissue matrix.
 11. The tissue sheet product of claim 1, wherein the first sheet of material is dry.
 12. The tissue sheet product of claim 1, wherein the first sheet of material is hydrated and flexible.
 13. The tissue sheet product of claim 1, wherein the plurality of elongated tissue matrix fragments are joined without use of substantial amounts of binder or adhesive.
 14. The tissue sheet product of claim 1, comprising a second sheet of material, wherein the second sheet of material is stacked on the first sheet of material such that the plurality of elongated tissue matrix fragments of the first sheet of material are oriented in a different direction relative to the plurality of elongated tissue matrix fragments of the second sheet of material.
 15. The tissue sheet product of claim 1, wherein an internal negative pressure is applied within the first sheet of material.
 16. The tissue sheet product of claim 1, comprising a second sheet of material layered over the first sheet of material and joined to the first sheet of material.
 17. A method for producing a tissue sheet product, comprising: treating a tissue matrix to produce a plurality of elongated tissue matrix fragments having a length between about 5 μm and 300 μm, at least a portion of the plurality of elongated tissue matrix fragments including frayed proximal and distal ends; forming the plurality of elongated tissue matrix fragments into a first sheet of material; and treating the first sheet of material to join the plurality of elongated tissue matrix fragments to one another at the frayed proximal and distal ends.
 18. The method of claim 17, further comprising drying the first sheet of material.
 19. The method of claim 18, wherein drying includes subjecting the first sheet of material to a convective drying process.
 20. The method of claim 18, wherein treating the first sheet of material to join the plurality of elongated tissue matrix fragments includes treating the first sheet of material with a dehydrothermal treatment process.
 21. The method of claim 17, wherein forming the plurality of elongated tissue matrix fragments into the first sheet of material includes compressing groups of the plurality of elongated tissue matrix fragments.
 22. The method of claim 17, wherein treating the tissue matrix to produce the plurality of elongated tissue matrix fragments includes milling the tissue matrix.
 23. The method of 17, wherein the plurality of tissue matrix fragments are strands of tissue matrix.
 24. The method of claim 17, wherein the tissue matrix is dermal tissue matrix.
 25. The method of claim 17, wherein the tissue matrix is porcine tissue matrix.
 26. The method of claim 17, wherein the tissue matrix is acellular tissue matrix.
 27. A method of treating a tissue site with a tissue sheet product, comprising: selecting a tissue sheet product, the tissue sheet product including a first sheet of material, wherein the first sheet of material comprises a plurality of elongated tissue matrix fragments having a length between 5 μm and 300 μm, wherein at least a portion of the plurality of elongated tissue matrix fragments include frayed proximal and distal ends, and wherein the plurality of elongated tissue matrix fragments are jointed to one another at the frayed proximal and distal ends to form the first sheet of material; and placing the tissue sheet product in or on the tissue site.
 28. The method of claim 27, further comprising removing tissue from the tissue site before placing the tissue sheet product in or on the tissue site.
 29. The method of claim 27, wherein the tissue site is a lumpectomy site.
 30. The method of claim 27, wherein the tissue site is selected from at least one of breast, a portion of an abdominal wall, a connective tissue, a tendon, a ligament, dermis, dura, cartilage, bone, gingiva, and adipose tissue.
 31. The method of claim 27, further comprising applying reduced pressure therapy to the tissue site.
 32. The method of claim 27, wherein the tissue matrix includes dermal tissue matrix.
 33. The method of claim 27, wherein the tissue matrix includes porcine tissue matrix.
 34. The method of claim 27, wherein the tissue matrix includes acellular tissue matrix. 